The acyl-CoA dehydrogenases (ACDs) are a family of highly conserved enzymes which catalyze the first intra-mitochondrial step in fatty acid beta-oxidation and branched chain amino acid metabolism. Deficiencies of these enzymes represent a cause of considerable morbidity and mortality in both children and adults and can present with episodic metabolic decompensation, mental retardation, myopathy, cardiomyopathy, hypoglycemia and peripheral neuropathy. Abnormalities of the short chain acyl-CoA dehydrogenase (SCAD) have proven particularly difficult to diagnose and study. Two common variants of SCAD have been identified, however, their role in causing disease is unclear. In the first funding period of this grant, we have shown that the two common polymorphic variants are functionally impaired, suggesting a possible pathophysiologic role in these individuals. We have also identified a new ACD [isobutyryl-CoA dehydrogenase (IBD)] active in the valine catabolic pathway, as well as the first patients deficient in IBD and short/branched chain ACD (SBCAD). The long range goal of the project continues to be characterization of the metabolism of short chain acyl-CoAs in humans, and deficiency of these enzymes at the biochemical, structural and molecular level. Specific aims of this renewal application include: Specific Aim 1, to identify molecular defects responsible for causing SBCAD, and IBD deficiencies, and to characterize the effects of mutations on enzyme function. Specific Aim la is to identify mutations in the SBCAD and IBD genes in patients with deficiencies of these enzymes. Specific Aim lb is to demonstrate the biological importance of SBCAD and IBD mutations using a variety of in vivo and in vitro expression techniques. Specific Aim lc is to further characterize genotype/phenotype relationships in these disorders. Specific Aim 2 is to characterize the structural motifs important in determining substrate specificity in the branched chain ACDs. Specific Aim 2a is to characterize substrate binding to rat and human SBCAD using surface plasmon resonance techniques. Specific Aim 2b is to determine the amino acid residues and motifs important in determining the specificity of SBCAD and IBD towards short and short branch chain acyl-CoA substrates. Specific Aim 2c is to determine the crystal structure of SBCAD and IBD. This will be performed by my collaborator Dr. Jung-Ja Kim. Specific Aim 3 is the determination of amino acid residues and motifs important for stabilization of ACD homotetramers. Aim 3a is to make mutant human IVDs based on the potato enzyme that will be stable, active dimers. Aim 3b is to identify residues in human IVD that are important in determining tetramer stability.